Injectable implants for tissue augmentation and restoration

This application is continuation of U.S. Ser. No. 09/258,723, filed on Feb. 26, 1999 now abandoned; which claims the benefit of U.S. Provisional Application No. 60/076,297, filed on Feb. 27, 1998 and U.S. Provisional Application No. 60/087,155, filed on May 29, 1998.

This invention was made with government support under Grant No. R43 HD34659-01 and Grant No. 1R43 HD34999-01 awarded by the National Institutes of Health, National Institute of Child Health and Human Development. The U.S. Government has certain rights in the invention as a result of this support.

The present invention relates to the field of polymeric materials that can be used as implants (injectable or otherwise insertable) in mammals for hard and soft tissue augmentation.

A number of methods exist for plastic or reconstructive surgery using injectable implants. The implants have been used for cosmetic reasons, such as filling in dermal creases, and for medical reasons, such as in the treatment of urinary incontinence. Concerning urinary incontinence, this medical condition affects 10 million, mostly elderly, Americans at a cost conservatively estimated at more than $10 billion annually. Clearly, the commercial applications of a successful material or family of materials for tissue augmentation, including urinary incontinence, would be substantial.

An additional example of the potential for soft tissue is seen in the area of intervertebral disc repair. Each year in the U. S. low back pain results in productivity losses that are greater than for any other medical condition and results in health care costs of more than $33 billion. When disability and lost productivity are added, the economic losses exceed $100 billion per year. The common cause of low back pain is pathology of a soft tissue, the intervertebral disc. When disc degeneration occurs, a collapse of the disc space occurs, which leads to neuroforaminal narrowing and nerve impingement. Soft tissue restoration or repair of an injured intervertebral disc could theoretically occur at two levels. One level is to improve the outcome of a discectomy or laminectomy procedure by using materials to prevent the adhesions and fibrosis that result in failed back surgery syndrome. Another level is to use a material to regain the correct disc dimensions and viscoelastic properties and at the same time to provide for cellular attachment where cells can sense the forces that an approximately configured disc would sustain. To date, satisfactory materials for these purposes have not been developed.

Among the materials that have previously received serious consideration as periurethral bulking agents to combat urinary incontinence arising from such conditions as intrinsic sphincter deficiency (ISD) are a synthetic organic polymer, polytetrafluoroethylene (PTFE) (Blaivas and Jacobs, J. Urol. 145:1214-1218 (1991); Malizia, et al., JAMA 251:3277-3281 (1984)), autologous fat (Santarosa and Blaivas, J. Urol. 151:607-611 (1994)), sodium morrhuate (Murless, J. Obstet. Gynaecol. 45:67-73 (1938)), and paraffin (Quackels, Acta. Urol. Belg. 23:259-262 (1955)). While the initial use of PTFE indicated a 73% improvement rate for stress urinary incontinence (Blaivas and Jacobs (1991)), distant particle migration to the lungs, liver, spleen and brain was subsequently observed with formation of foreign body granulomas (Malizia, et al., supra). Other materials have been reviewed by Canning ( Dial. Ped. Urol. 14 (1991)). Injectable bioglass has also been considered, but a 16- to 18-gauge needle appears to be required for injection, which results in tissue damage and leakage of the bioglass (Walker, et al., J. Urol. 148:645-647 (1992)).

Collagen, a natural component of connective tissue, has also been used for soft tissue augmentation (U.S. Pat. No. 5,428,022; Richardson, et al., Adult Urology, 46:378-381 (1995); Frank, et al., Plastic and Reconstructive Surgery 87:1080-1088 (1991); WO95/26761), as have polymer conjugates, such as polyethyleneglycol (U.S. Pat. No. 5,476,666). A glyceraldehyde cross-linked bovine dermal collagen with reduced antigenicity and increased resistance to fibroblast-secreted collagenases emerged as a promising material with about 80% of treated patients being either cured or improved after injection with this material (Richardson, et al. (1995); Stricker and Haylen, The Medical Journal of Australia, 158:89-91 (1993)). However, recent studies have shown that the cure rate is actually 25%, with 46% of the successful cases needing repeated injections within 3 years, i.e., a cure rate of 10-15% at the 3 year mark. Herschorn, et al., J. Urol. 156:1305-1309 (1996). In addition, up to 5% of patients exhibit a hypersensitivity reaction following the required intradermal skin test with this material (Siegle, et al., Arch. Dermatol. 120:183-187 (1984)), and are thus not suitable candidates for collagen therapy. The injection results in a mild inflammatory response in which the injected collagen attracts nearly equal amounts of host collagen over a period of about six months, resulting in permanent scarring (Stricker and Haylen (1993)). Such scarring can complicate further efforts at treatment. Furthermore, the material is completely degraded in 9 to 19 months, resulting in a need for repeated injections (Appell, The Craft of Urological Surgery 21(1):177-182 (1994)).

Artificial bioelastic polypeptides are a relatively new development that arose in the laboratories of the present inventor and are disclosed in a series of previously filed patents and patent applications. For example, U.S. Pat. No. 4,474,851 describes a number of tetrapeptide and pentapeptide repeating units that can be used to form a bioelastic polymer. Specific bioelastic polymers are also described in U.S. Pat. Nos. 4,132,746; 4,187,852; 4,589,882; and 4,870,055. U.S. Pat. No. 5,064,430 describes polynonapeptide bioelastomers. Bioelastic polymers are also disclosed in related patents directed to polymers containing peptide repeating units that are prepared for other purposes but which can also contain bioelastic segments in the final polymer: U.S. Pat. Nos. 4,605,413; 4,976,734; and 4,693,718, entitled “Stimulation of Chemotaxis by Chemotactic Peptides”; U.S. Pat. No. 4,898,926, entitled “Bioelastomer Containing Tetra/Pentapeptide Units”; U.S. Pat. No. 4,783,523 entitled “Temperature Correlated Force and Structure Development of Elastin Polytetrapeptide”; U.S. Pat. No. 4,500,700, entitled “Elastomeric Composite Material Comprising a Polypeptide”; U.S. Pat. No. 5,250,516 entitled “Bioelastomeric Materials Suitable for the Protection of Wound Repair Sites”; U.S. Pat. No. 5,527,610 entitled “Elastomeric Polypeptide Matrices for Preventing Adhesion of Biological Materials”; and U.S. Pat. No. 5,336,256 entitled “Elastomeric Polypeptides as Vascular Prosthetic Materials”.

A number of other bioelastic materials and methods for their use are described in pending U.S. patent applications, including: U.S. Ser. No. 08/316,802, filed Oct. 3, 1994, entitled “Bioelastomeric Drug Delivery System”; U.S. Ser. No. 08/187,441, filed Jan. 24, 1994, entitled “Photoresponsive Polymers”; U.S. Ser. No. 08/487,594, filed Jun. 7, 1995, entitled “Polymers Responsive to Electrical Energy” and published as PCT/US96/09776; U.S. Ser. No. 08/735,692, filed Oct. 16, 1995 entitled “Bioelastomers Suitable as Food Product Additives” and published as PCT/US96/05266; U.S. Ser. No. 08/542,051 filed Oct. 13, 1995, entitled “Hyperexpression of Bioelastic Polypeptides”; U.S. Ser. No. 08/543,020 filed Oct. 13, 1995, entitled “A Simple Method for the Purification of a Bioelastic Polymer” and published as PCT/US96/05186. All of the aforementioned patents and patent applications are herein incorporated by reference, as they describe in detail bioelastomers and/or components thereof and their preparation.

Artificial bioelastic materials are based on elastomeric and related polypeptides comprised of repeating peptide sequences (Urry, Angew. Chem. (German) 105:859-883 (1993); Angew. Chem. Omt. Ed. Engl. 32:819-841 (1993)). As a result of work conducted by the present inventor, the bioelastic polypeptides based on VPGVG have been found to be soluble in water below 25° C., but on raising the temperature they associate reversibly to form a dense, water-containing viscoelastic phase in the polypentapeptide (“PPP”) and polytetrapeptide (“PTP”) cases, whereas the polyhexapeptide (“PHP”) associates irreversibly in water to form a granular precipitate, which usually requires the addition of trifluoroethanol to the aggregate for redissolution. The viscoelastic phase is called the coacervate, and the solution above the coacervate is referred to as the equilibrium solution. On cross-linking, the PPP and PTP polymers have been found to be elastomers, whereas the PHP polymer is not elastomeric and appears to provide a means for aligning and interlocking the chains during elastogenesis.

The process of raising the temperature to form the elastomeric state is an inverse temperature transition resulting in the development of a regular dynamic structure, unlike the random network structure of typical rubbers. The regular structure is a β-spiral, a loose water-containing helical structure with β-turns and spacers between turns of the helix which provides hydrophobic contacts between helical turns and has suspended peptide segments. These peptide segments are free to undergo large amplitude, low frequency rocking motions called librations. In addition, several of these β-spirals associate to form twisted filaments. The elastomeric force of these various bioelastomers increases as the regular dynamic structure thereof develops. By synthesizing bioelastic materials having varying mole fraction amounts of the constituent repeating units and by choosing a particular solvent to support the initial viscoelastic phase, it is possible to rigorously control the temperature at which the obtained bioelastomer develops elastomeric force. Maximum elastomeric force develops over a relatively narrow temperature range at temperatures spanning a range of up to about 75° C.

Bioelastic materials have been proposed for a number of uses and apparatuses, as indicated by the general subject matter of the applications and patents set forth above, and have been made available in different physical forms, such as sheets, gels, foams, or powders. For example, compositions can be used in medical applications ranging from the prevention of post-surgical adhesions (Urry, et al., “Biotechnological Polymers: Medical, Pharmaceutical and Industrial Applications”, pp. 82-103 (1993)) to programmed drug delivery. See also Urry, “Bioelastic Materials as Matrices for Tissue Reconstruction,” in Tissue Engineering Current Perspectives , (Eugene Bell. Ed.), Birkhauser Boston, Div. Springer-Verlag, New York, N.Y., pp. 199-206 (1993). Materials functioning as insulator materials for isolating wound repair sites from adhesions, for the protection of burn areas, and to facilitate repair of the damaged tissue have been described in U.S. Pat. Nos. 5,250,516 and 5,527,610.

Despite the numerous techniques that have existed in the past for tissue augmentation, all of the existing techniques have suffered from one deficiency or another that have resulted in less than optimal results for the patient. There remains a need for biocompatible implants, especially injectable implants, for tissue augmentation, and the present invention provides compositions and methods for meeting those needs that do not suffer from the deficiencies of prior techniques.

It is an object of the invention to provide long-lasting implants for tissue augmentation or restoration.

It is also an object to provide implants that are injectable under a variety of surgical conditions, including emplacement during endoscopic surgery and related techniques.

It is a further object to provide for implants that match the compliance of the tissue site of application and that can be selected to match different compliance values with a minimum of difficulty.

It is yet another object to provide an implant that has both a carrier polymer that is biologically inert or at least degradable to non-toxic products, and a biologically active component.

It is another object of the invention to provide an implant that is readily sterilizable as well as being biocompatible, eliciting insignificant immunogenic and antigenic responses in the host.

It is a further object of the invention to provide an implant that, in biological situations involving implantation of exogenous material into a tissue, as in tissue regeneration or restoration, can stimulate cell adhesion and resulting cell growth upon appropriate modification or additions to the basic structure of the composition.

It is yet another object of the invention to provide a method for tissue augmentation, such as in plastic surgery (e.g., wrinkle removal) or to correct medical conditions (e.g., urinary incontinence or back pain resulting from degenerative defects in intervertebral discs).

These and other objects of the invention are achieved by providing a method for tissue augmentation in a warm-blooded animal comprising injecting a polymer at the tissue site in need of augmentation, which site has a tissue temperature, said polymer comprising repeating peptide monomeric units selected from the group consisting of nonapeptide, pentapeptide and tetrapeptide monomeric units, wherein said monomeric units form a series of β-turns separated by dynamic bridging segments suspended between said β-turns, wherein said polymer has an inverse temperature transition T t less than said tissue temperature, and wherein said polymer is injected as a water solution at coacervate concentration in the substantial absence of additional water. The monomeric units can all be the same or they may be different. In specific embodiments, tissue reconstruction can be advantageously achieved using a polymer having the same elastic modulus of the natural tissue being augmented and, in some case, having the capability to induce natural cells of the tissue (or surrounding tissue) to enter and to function at the site of reconstruction.

Further objects of the invention are achieved by providing a method for tissue restoration of intervertebral discs in a mammal, said method comprising: injecting a polymer into the depleted nucleus pulposus site, which has a site temperature, said polymer comprising repeating peptide monomeric units selected from the group consisting of nonapeptide, pentapeptide and tetrapeptide monomeric units, wherein said monomeric units form a series of β-turns separated by dynamic bridging segments suspended between said β-turns, wherein said polymer has an inverse temperature transition T t less than said site temperature, and wherein said polymer is injected as a water solution at coacervate concentration in the substantial absence of additional water and swells to increase the pressure within the disc.

The injectable protein-based polymers as taught herein can be designed to have numerous advantages including biological stability, biological function, and defined polymer size. These advantages are achieved by providing polymers composed of easily obtained and coupled monomer units, e.g. amino acids, that are themselves diverse in structure and in chemical properties and are readily modified. Furthermore, recombinant peptide-engineering techniques can be advantageously used to produce specific peptide backbones, either in bioelastic units or non-elastic biofunctional segments. Thus, the polymer can be present as a copolymer containing a mixture of tetrameric, pentameric or other monomeric units.

The polymers can be prepared with widely different water compositions, with a wide range of hydrophobicities, with almost any desired elastic modulus, and with a variable degree of cross-linking by selecting different amino acids for the different positions of the monomeric units and by varying the cross-linking process (e.g. chemical, enzymatic, or radiation) used to form the final product. Preparation of a variety of polymers that can be used in the present invention has already been described, for example in the publications discussed above, and the preparation of the polymers themselves is now established and not a novel part of the present invention, although the preparation of these materials as injectable formulations for use in tissue augmentation is new. Additionally, there are some preferred embodiments of polymers that can be prepared as described herein that are themselves new. For the most part, however, the invention involves the application of existing polymers (as well as new polymers) to a new field where they have not been used before.

The invention will be better understood by reference to the following detailed description of specific embodiments together with the figures that form part of this specification, wherein:

FIG. 1 illustrates an oligonucleotide sequence (SEQ ID NO:1) encoding the bioelastic polymer protein sequence made up of the repeating [(GGVP) 3 GGFP (GGVP) 3 GGFP (GGVP) 3 GGFP] (SEQ ID NO:9) unit, and shows the encoded protein sequence and restriction enzyme sites.

FIG. 2 illustrates an oligonucleotide sequence (SEQ ID NO:2) encoding the bioelastic polymer protein sequence (GVGVP) 2 GKGVP GVGVP GVGFP GFGFP (SEQ ID NO: 10) and restriction enzyme sites.

FIG. 3 illustrates an oligonucleotide sequence (SEQ ID NO:3 and 4) encoding the bioelastic polymer protein sequence VGVP GRGDSP G (SEQ ID NO:11).

FIG. 4 illustrates an oligonucleotide sequence (SEQ ID NO:5) encoding the bioelastic polymer protein sequence (GVGVP) 3 GKGVP (GVGVP) 2 (SEQ ID NO: 12).

FIG. 5 illustrates an oligonucleotide sequence (SEQ ID NO:6) encoding the bioelastic polymer protein sequence (GVGVP) 10 GVGVPGRGDSP (GVGVP) 10 (SEQ ID NO: 13).

FIG. 6 illustrates an oligonucleotide sequence (SEQ ID NO:7) encoding the bioelastic polymer protein sequence (GVGVP) 10 (GVGVAP) 8 (GVGVP) 10 (SEQ ID NO:14).

FIG. 7 illustrates an oligonucleotide sequence (SEQ ID NO:8) encoding the bioelastic polymer protein sequence (GVGVP) 3 GEGVP (GVGVP) 2 (SEQ ID NO:15).

FIG. 8 illustrates the sequence of oligonucleotide adapters useful in cloning bioelastic polymer genes and are identified as SEQ ID NOS:30, 31, 32 and 33.

FIG. 9 is a mass spectra plot of polymers III and IV.

FIG. 10 illustrates the sequence of oligonucleotide adapters useful in cloning bioelastic polymer genes and are identified as SEQ ID NOS:63 and 64.

The process of soft tissue augmentation has typically been achieved in the past using synthetic polymers or collagen derivatives. The prior art methods involving synthetic polymers, such as PTFE (polytetrafluoroethylene), have distinct disadvantages in that synthetic polymers are recognized by the body as foreign material and PTFE promotes granuloma formation. Although collagen derivatives have been used with some success, they need to be injected with excess water, resulting in complications arising from the subsequent decrease in filling volume. In addition, collagen derivatives degrade and promote scar tissue formation over time. These and other disadvantages of the prior art materials used in tissue augmentation are overcome in the present invention by the use of injectable bioelastic polymers as implants. The methods of collagen injection, however, can in the most part be applied directly to methods of using the present compositions for tissue augmentation, subject to the variations described herein.

The defined structure of the artificial bioelastic polymers used in the invention allow polymers to be designed and synthesized with chosen physical properties, rather than having to rely on the less controllable properties of materials prepared from natural products. It should be noted that the terms “bioelastic polymers”, “bioelastomeric polymers,” and “bioelastomers” are used interchangeably herein and further that these terms encompass materials that may not be thought of as elastomers (such as certain polymers having the characteristics of plastics), since the term “bioelastomer” has come to be an art-recognized term describing polymers comprising repeating peptide monomeric units selected from the group consisting of nonapeptide, pentapeptide and tetrapeptide monomeric units, where the monomeric units form a series of β-turns separated by dynamic bridging segments suspended between the β-turns. Examples of these materials (in their prior embodiments) are described in the numerous publications, including patents, that are listed above and that are incorporated herein by reference.

The bioelastomers can be described either generically, as above, or with various degrees of specificity. The generic descriptions have been sufficient for the inclusion in claims of a number of issued U.S. and foreign patents. However, a number of subgeneric and specific descriptions will be provided for those less familiar with bioelastomers.

One is to describe a grouping of these materials that contain ionizable amino acid residues (used for purposes described below) as containing repeating units of the formula αPρΩG or VPθδ, wherein:

V is a peptide-forming residue of L-valine;

P is a peptide-forming residue of L-proline;

G is a peptide-forming residue of glycine;

α is a peptide-forming residue of L-valine, L-leucine, L-isoleucine, L-phenylalanine or an ionizable peptide-forming residue selected from the group consisting of the residues of L-Glu, L-Asp, L-His, L-Lys, L-Tyr, and other ionizable peptide-forming L-amino acids;

ρ is a peptide-forming residue of glycine or a peptide-forming residue of D-Glu, D-Asp, D-His, D-Lys, D-Tyr, or (optionally) other ionizable peptide-forming D-amino acids for the elastic polymeric repeats or any L-amino acid for the elastic forming repeats;

θ is a peptide-forming residue of L-valine, L-leucine, L-isoleucine, L-phenylalanine or (optionally) an ionizable peptide-forming L-amino acids;

Ω is a peptide-forming residue of glycine or a peptide-forming residue of D-Glu, D-Asp, D-His, D-Lys, D-Tyr, or (optionally) another ionizable peptide-forming D-amino acid; and

δ is a peptide-forming residue of glycine or a peptide-forming residue of L-Glu, L-Asp, L-His, L-Lys, L-Tyr, or (optionally) another ionizable peptide-forming L-amino acid.

More generally, bioelastic polypeptide polymers generally comprise repeating elastomeric units selected from the group consisting of bioelastic pentapeptides and tetrapeptides, where the repeating units comprise amino acid residues selected from the group consisting of hydrophobic amino acid and glycine residues and where the repeating units exist in a conformation having a β-turn of the formula:

wherein R 1 -R 5 represent side chains of amino acid residues 1-5, and m is 0 when the repeating unit is a tetrapeptide or 1 when the repeating unit is a pentapeptide. Nonapeptide repeating units generally consist of sequential tetra- and pentapeptides. Preferred hydrophobic amino acid residues are selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. In many cases, the first amino acid residue of the repeating unit is a residue of valine, leucine, isoleucine or phenylalanine; the second amino acid residue is a residue of proline; the third amino acid residue is a residue of glycine; and the fourth amino acid residue is glycine or a very hydrophobic residue such as tryptophan, phenylalanine or tyrosine.

A particularly useful tetrapeptide is Val-Pro-Gly-Gly (SEQ ID NO:16). Also useful are GGVP (SEQ ID NO:41), GGFP (SEQ ID NO:42) and GGAP (SEQ ID NO:50). A particularly useful pentapeptide is Val-Pro-Gly-Val-Gly (SEQ ID NO:17). Also useful are GVGVP (SEQ ID NO:20), GKGVP (SEQ ID NO:43), GVGFP (SEQ ID NO:44), GFGFP (SEQ ID NO:45), GEGVP (SEQ ID NO:48), GFGVP (SEQ ID NO:49) and GVGIP (SEQ ID NO:51). For other specifically preferred individual monomeric units and bioelastomers, see any of the patents that are herein incorporated by reference.

The bioelastomers of the invention can consist of only nonamers (a polynonapeptide), tetramers (a polytetrapedptide), only pentamers (a polypentapeptide) or a mixture of these units, but more typically a mixture of tetrapeptide and pentapeptide units (a copolymer). In addition, the bioelastomer can be a copolymer formed from one of the aforementioned monomeric units and a second peptide unit containing 1-100 amino acids, more typically 1-20 amino acids. On the smaller side, this second peptide can be for example, the fibronectin cell attachment sequence, GRGDSP (SEQ ID NO:46) or a monomer such as GVGVAP (SEQ ID NO:47) or VGVAPG (SEQ ID NO:52), which is a chemoattractant for fibroblasts and monocytes. On the larger side (90-100 amino acids), the second peptide can be a cell attachment sequence from the Type-III domains of fibronectin, vitronectin, tenascin, titin or other related cell attachment protein, which sequence provides more specific cell attachment than the somewhat non-specific GRGDSP cell attachment sequence.

In one aspect, the present invention provides injectable bioelastic polymers and methods for their preparation and use, where the polymers are selected for and exhibit specific desired properties for tissue augmentation. For example, the polymers preferably exhibit biocompatibility, extrusibility through a fine surgical needle (e.g., 22-gauge) at the temperature of use, as well as insignificant volume change after injection. The later characteristic is important, since the preferred coaptation (extent of filling) at the treated site can be assessed visually at the time of injection, for example, by cystoscopical examination, without the need to consider subsequent increases or decreases in filling volume and related complications. The bioelastic polymers can also be designed to remain at the site of injection, and to provide long-lasting tissue augmentation, as is described in more detail below.

In another aspect, the present invention provides injectable bioelastic polymers and methods for their preparation and use, where the polymers are selected for and exhibit specific desired properties for tissue restoration in the area of intervertebral disc repair. For example, as for tissue augmentation applications, the polymers preferably exhibit biocompatibility. However, for disc repair, the polymers are more viscous at the temperature of use and are injected through a larger bore such as is present in equipment used in endoscopic or percutaneous discectomy procedures, as well as exhibiting a significant volume change after injection by swelling. Certain polymers are preferred for this application, especially those polymers containing one or more pentapeptide units such as GVGIP (SEQ ID NO:51) or one or more monomers that contain one or more, typically one or two, aromatic residues such as phenylalanine, for example, GVGFP (SEQ ID NO:44), GFGFP (SEQ ID NO:45), and GFGVP (SEQ ID NO:49).

“Injectable” as used herein relates to materials having a texture and viscosity which permits their flow through a suitable surgical needle or other surgical instrument, such as a equipment used in endoscopic or percutaneous discectomy procedures, by employing typical injection pressures. For example, an injectable material of low viscosity can be forced through at least the diameter of a 22-gauge needle. Injectable materials of high viscosity can be introduced, for example, by first making a hole in the annulus with a trocar, then forcing the bioelastomer through the bore of large syringe, i.e., a syringe of about 13 to 19-gauge, under normal pressure applicable by the hand and fingers of a surgeon. The mixture is injected directly into the site in need of augmentation, such as dermis (subdermal), intervertebral disc, periurethral, tendon or cartilage, and causes essentially no detectable inflammation or foreign body reaction.

“Augmentation” means the repair, prevention, or alleviation of defects, particularly defects due to loss or absence of tissue, by providing, augmenting, or replacing such tissue. While the invention is primarily designed for soft tissue augmentation, hard tissue augmentation is encompassed as well since the injectable compositions of the invention can be used in combination with, for example, materials to promote mineralization or bone formation, as described elsewhere.

In its broadest aspect, the present invention provides a method for tissue augmentation in a warm-blooded animal by injecting a bioelastic polymer at a tissue site in need of augmentation. The method will often be applied to humans, but one can also readily understand that the present method is applicable to agriculturally important animals, including mammals, such as cattle, horses, pigs, and sheep, and other animals such as chickens, turkeys, and geese (as for example in providing for repair of breeding stock that has suffered a traumatic injury), as well as domestic animals of interest, such as cats and dogs.

In another embodiment of the invention, the method for tissue augmentation or restoration in a mammal, comprises the steps of a) identifying a tissue site in need of tissue augmentation or restoration, such site having a site temperature (T s ); and b) injecting a polymer at the site, said polymer comprising repeating peptide monomeric units selected from the group consisting of pentapeptide and tetrapeptide monomeric units, alone or in combination, wherein the monomeric units form a series of β-turns separated by dynamic bridging segments suspended between the β-turns, and wherein (i) the polymer has an inverse temperature transition T t less than T s , (ii) the polymer is injected as a water solution at coacervate concentration in the substantial absence of additional water, and (iii) the coacervate has a viscosity at T s of 1 to 100,000 millipoise.

Bioelastic polymers are high molecular weight polymers of repeating peptide sequences and are the subject of numerous patents and patent applications from the laboratories of the present inventor, as described in the Background Section. Bioelastomers exhibit inverse temperature transitions in the form of a phase separation in which folding and aggregation of water-soluble polymer chains into more-ordered states of the condensed (coacervate) phase occur on raising the temperature. Although the prior patents and applications have not been concerned with injectable compositions for tissue augmentation, they provide considerable guidance on biocompatibility and on manufacturing of bioelastomers to obtain useful structural features for the uses described herein.

Particularly preferred bioelastic materials are those based on elastomeric tetrapeptide, pentapeptide, and nonapeptide monomers or monomeric units, as these materials have already been demonstrated to be biocompatible. The elasticity of the monomeric units is believed to be due to a series of β-turns in the protein's secondary structure, i.e., the conformation of its peptide chain separated by dynamic (as opposed to rigid) bridging segments suspended between the β-turns. Typical polymers contain at least 5, preferably at least 10, more preferably at least 20 monomers, and because of limited solubility in aqueous solvents, which are desirable for biological uses, usually contain fewer than 1000, usually fewer than 500, of such units.

Injectable bioelastic polymers are soluble in water at all proportions at a sufficiently low temperature, but on raising the temperature above a critical value, designated as T t , they fold, assemble and phase separate into a more ordered state of predetermined composition, such as on the order of 50% peptide/50% water by weight. However, this value does vary and a broad range is acceptable for use in the applications of the invention. For example, polymers containing the pentapeptide monomer, GVGVP, have been shown to have a resultant ordered state of 40 wt % peptide/60 wt % water, while polymers containing GVGIP have a resultant ordered state of 60 wt % peptide/40 wt % water. This phase separation is referred to as an inverse temperature transition and the more-dense phase is called a coacervate.

The transition temperature of the particular bioelastic polymer is preferably selected to be below the normal temperature of the tissue in which the composition will be injected, which will vary slightly with the location of the tissue, as is well known. Skin, for example, in humans has a slightly lower temperature than internal tissues. Thus, most of the time T t will be selected to be less than 37° C., preferably below 35° C., more preferably below 30° C., even more preferably below 25° C. and most preferably below 20° C. to provide a stable coacervate. Preparation of a bioelastomer with a pre-selected T t of 25° C. has been previously described on numerous occasions. Table I delineates the T t -based hydrophobicity scale which may be used to change an otherwise non-functional residue to return the transition temperature where desired. Thus, Table 1 represents a scale for protein engineering.

Virtually every variable can, with the appropriate composition of the protein-based polymer, change the value of T t . Such variables include, among other criteria, (1) polymer concentration, (2) polymer length, (3) amino acid composition, (4) presence of salts e.g. the Hofmeister (Lyotropic) Series, (5) organic solutes and solvents, (6) polymer side-chain ionization, (7) chemical modification of polymer side-chains e.g. phosphorylation or nitration, (8) pressure e.g., as effecting aromatic residues, (9) redox state of chemical groups attached to the polymer, (10) light absorption by chemical groups attached to the polymer, and (11) side chain neutralization by ion pairing e.g., cation neutralization of anionic side chains, anion neutralization of cationic side chains, and ion-pairing between side chains.

Bioelastomers for use in the practice of the invention can be readily prepared at an appropriate viscosity, although this has not previously been of interest in the preparation of these materials. Typically, the coacervate has a viscosity at the tissue site temperature of 1 to 100,000 millipoise. By selection of the monomers used, the length of the polymer chains, and the amount of crosslinking (as is well known in the polymer art), viscosity can be readily controlled. In fact, a wide range of viscosity can be used depending upon the particular application. For example, it is desirable to have the viscosity of the coacervate sufficiently low to allow reasonable control of syringe being used by a physician or other individual to inject the polymer into the desired tissue location for tissue augmentation. On the other hand, for tissue restoration such as in intervertebral disc restoration, a higher viscosity is desired when the polymer is to be injected through a larger syringe.

The injectability of the polymer is usually controlled by the viscosity of the coacervate concentration, which is the form in which injection usually takes place. Measurement of viscosity is not required, however, as suitability can be readily assessed by simply attempting to extrude any previously unused composition from the syringe to be used through a needle of the appropriate size. For example, the polypentapeptide (GVGVP) 251 (SEQ ID NO: 18) is extrusible through a 22 and even a 27 gauge needle at 37° C., after having been initially dissolved at 4° C. at a concentration of 500 mg/ml (and after expelling of excess water from the coacervate, as described below). Preferred bioelastic materials for use in the tissue augmentation and restoration aspects of the present invention include polytetrapeptides, such as poly[0.6(GGVP),0.4(GGFP)] (SEQ ID NOS:41 and 42), which are extrusible through both 22 and 27 gauge needles at 37° C. and remain extrusible for sufficient time (weeks to months) to allow storage of the polymer in ready-to-use form. Preferred bioelastic materials for use in the restoration of intervertebral discs will have thicker viscosities, typically exhibiting an elastic modulus within the range of 5×10 4 to 5×10 6 N/m 2 at the temperature at the disc site. It may be desirable to approximate the elastic modulus of the vascular wall of the femoral artery, which is about 5×10 5 N/m 2 , and even more preferable to approximate the elastic modulus of the nucleus pulposus, which is less than 5×10 5 N/m 2 . Although particular polymer systems are mentioned illustratively here and elsewhere, it will be recognized that such a description is not limiting of the invention, as these and other examples can be readily modified to provide numerous compositions that have the desired properties for tissue augmentation as discussed herein.

Polymers are injected in the method of the invention in a coacervate composition from which all, or substantially all, of the excess water has been excluded. As previously noted, the coacervate state of the polymers used in this method is essentially a viscoelastic state containing a fixed ratio of water and polymer. Excess water present from the initial dissolution of polymer into water at a temperature below T t, usually 4° C., is readily removed from this solid phase by any of the techniques available for separating phase separated states from supernatant water. The coacervate therefore provides a stable volume under physiological conditions, allowing a polymer to be injected from a suitable surgical needle without being subject to volume changes at the injection site after injection, as occurs with injections of collagen, which require the presence of excess water. A plastic surgeon can therefore readily assess the amount of polymer to be injected by observation without having to estimate later shrinkage of the injection site caused by water leakage from the site.

It will of course be recognized that some amount of excess water can remain with the coacervate without detracting from the practice of the invention, as long as the amount is not sufficient to impair the injection of the coacervate. For example, as much as 30% (by volume) excess water can be present if the surgeon is willing to make adjustments in the injected volume to anticipate the final volume, although no more than 20% excess water is preferred, more preferably no more than 10%, and most preferably no more than 5.

As with all bioelastic polymers, transition to the coacervate state can occur without a change in temperature but under the control of other environmental factors. This is referred to as the ΔT t mechanism of transition (U.S. Ser. No. 08/187,441; Urry et al., Biopolymers 24:2345-2346 (1985), herein incorporated by reference) and can be achieved by changing a number of intrinsic or extrinsic factors. Intrinsic to a class of model proteins of 50,000 Da molecular weight or greater are the following: (a) the concentration of polymer itself, (b) changes in the amino acid composition within the polymeric bioelastic unit, (c) changes in the degree of ionization of functional side chains controlled by changes in pH, (d) the phosphorylation of side chains such as serine by enzymes called kinases, (e) the oxidation or reduction electrically, chemically or enzymatically of a side chain attached to the polymer, and (f) chemical reactions of side chains, for example in response to electromagnetic radiation. Extrinsic chemical changes affecting T t include the effects of salts, organic solutes and solvents. In addition, there is a chain length dependence that becomes significant at lower molecular weights where a polymer having shorter peptide chain lengths exhibit higher values of T t relative to a polymer having longer peptide chain lengths.

Thus, bioelastomers can be rationally designed in order to achieve the desired properties appropriate for the methods of the invention. The choice of individual amino acids from which to synthesize the elastomeric units and resulting polypeptide is unrestricted so long as the resulting structure comprises elastomeric structures with features described, for example, in U.S. Pat. Nos. 4,474,851 and 5,064,430, herein incorporated by reference, particularly β-turn formation and the resulting polymer maintains attributes useful for purposes intended according to the embodiments of the invention including, among other things, extrusibility through fine gage needles.

In general, selection of the sequence of amino acids in a particular monomeric unit and selection of the required proportion of monomeric units can be accomplished by an empirical process that begins with determining (or looking up) the properties of known bioelastomers, making similar but different bioelastomers and measuring the transition temperature and physical properties as described herein and in the cited patents and patent applications. The effect of changing the amino acid composition on the value of T t can also be determined using a hydrophobicity scale described in detail in U.S. patent application Ser. No. 08/187,441. For example, a rough estimate of the likely transition temperature can be obtained by summing the mean hydrophobicities of the individual amino acid residues in the monomeric units of the polymer and comparing the result to the sum obtained for polymers having known transition temperatures. Typically, more hydrophobic residues (e.g., Ile, Phe) lower T t , whereas less hydrophobic residues (e.g., Ala, Gly) and polar residues (e.g., Asp, Lys) raise T t .

Any chemical means of changing the mean hydrophobicity of the polymer, such as dephosphorylation/phosphorylation, reduction/oxidation of a redox couple, ionization/deionization, protonation/deprotonation, cleavage/ligation, amidation/deamidation, a conformational or a configurational change (e.g., cis-trans isomerization), an electrochemical change (e.g., pKa shift), emission/absorbance, or other physical change (e.g., heat energy radiation/absorbance), or pressure, or combinations thereof can be used to bring about transition. U.S. Pat. No. 5,226,292 from the laboratory of the present inventor details pressure-related effects, U.S. Ser. No. 08/187,441, photoresponsive effects, and U.S. Ser. No. 08/487,594, electroresponsive effects, each of which can be used to effect transition.

A major advantage of the bioelastic polypeptides is the extent to which fine-tuning of the degree of hydrophobicity/polarity and resulting shift in the inverse temperature transition can be achieved. For example, the hydrophobicity of the overall polymer (and therefore the average hydrophobicity of functional groups present in the polymer) can be modified by changing the ratio of different types of monomeric unit. These can be monomeric units containing a functional group undergoing the transition or other monomeric units present in the polymer. For example, if the basic monomeric unit is VPGVG (SEQ ID NO:17) and the unit undergoing transition is VPGXG (SEQ ID NO:19); where X is an amino acid residue modified to have an electroresponsive side chain, either the ratio of VPGVG unit to VPGXG units can be varied or a different structural unit, such as IPGVG, can be included in varied amounts until the appropriate transitions temperature is achieved. Furthermore the regularity of structure of the protein and protein-based bioelastic polymers allows optimal arrangement of the structural components. For example, optimal spatial proximity can be achieved by placing coupled residues adjacent to each other in the backbone (i.e., based on primary sequence) and also by positioning to provide inter-turn proximity.

The bioelastic polymers are composed of peptide units that form a matrix which can be modified in a variety of ways to obtain additional properties. For example, one or more of the peptide bonds can be optionally replaced by substitute linkages such as those obtained by reduction or elimination. Thus, one or more of the —CONH— peptide linkages can be replaced with other types of linkages such as —CH 2 NH—, —CH 2 S—, CH 2 CH—, —CH═CH— (cis and trans), —COCH 2 —, —CH(OH)CH 2 —, and —CH 2 SO—, by methods known in the art, for example, see Spatola, A. F. (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins (B. Weinstein, ed.) Marcel Dekker, New York, p. 267 for a general review. Amino acid residues are preferred constituents of these polymer backbones. Of course, if backbone modification is made in the elastomeric units, then suitable backbone modifications are those in which the elasticity and inverse temperature transition of the polymer is maintained.

As is apparent to one of ordinary skill in the art, the physical properties of the bioelastic polymer can be adjusted as described above, to exhibit desired characteristics, for example, viscosity, consistency, modulus of elasticity, stability, and the like. For example, the parent elastic protein-based polymer (GVGVP) n (SEQ ID NO:20), when prepared with n on the order of 200 and when cross-linked with 20 Mrads of γ-irradiation (described below), forms an elastic matrix with an elastic modulus approximating that of natural vascular wall, in the range of 10 5 N/m 2 . By variations in composition and conditions, the elastic modulus can be varied from 10 4 to 10 8 N/m 2 . This provides the capacity to match compliance over the wide range of biological soft tissues. In other embodiments, the primary sequence of the polypeptide can be designed to elicit fibrous capsule formation thus maintaining the polymer at the site of injection. A particular polymer can be assessed for its ability to promote fibrous capsule formation by methods known in the art, e.g., visually after a period of implantation in a test subject. In a preferred embodiment, an illustrative polymer that does not elicit fibrous capsule formation is X 20 -poly(GVGVP) (Urry, et al., J. Bioactive Compatible Polym. 6:263-282 (1991)). Polymers described in this specification that are prepared by irradiation cross-linking are identified as, for example, “X 20 -polyVPGVP,” which refers to a polymer prepared from VPGVG pentapeptide units which has been irradiated with a 20 Mrad dose of cobalt-60 radiation to form the cross-links, thus resulting in an insoluble matrix. Injectable, cross-linked coacervates would be obtained by much lower radiation dosages, usually less than 20 Mrad, often less than 10 Mrad, and even less than 5 Mrad.

In another preferred embodiment, a polymer that comprises phenylalanine (Phe, F) residues, such as X 20 -poly[0.75(GVGVP), 0.25(GFGVP)] (SEQ ID NOS:20 and 49) promotes fibrous capsule formation after implantation without the development of inflammation. Similarly, in another preferred embodiment subcutaneous injections of poly[0.6(GGVP), 0.4(GGFP)] (SEQ ID NOS:41 and 42) elicit fibrous capsule formation. Example 1 below illustrates such a polymer with (GGVP) 3 GGFP (GGVP) 3 GGFP (GGVP) 3 GGFP (SEQ ID NO:9).

Polymer characteristics can also be affected by cross-linking. Thus, the polymers used in the present invention can be cross-linked prior to, during, or after injection, if desired, using any of various cross-linking processes, e.g., chemical, enzymatic, or irradiative. Cross-linking provides mechanical strength and rigidity to the polymer, and increasing amounts of cross-linking are appropriate for increasing demands of rigidity. Although the most preferred polymers have little or no crosslinking prior to injection, cross-linking to provide one cross-link for every 200-500 repeating units is generally acceptable, with more cross-linking being permitted in less viscous polymers and vice versa. Methods for cross-linking bioelastomeric polypeptides are known in the art. For example, U.S. Pat. No. 4,589,882, incorporated herein by reference, teaches enzymatic cross-linking by synthesizing block polymers having enzymatically cross-linkable units. Additionally, cross-linking by irradiation is described in detail in nearly all of the prior patents arising from the laboratories of the present inventor.

In situ cross-linking can also be used after injection to maintain the polymer at the site of injection (Kagan, et al., J. Biol. Chem. 255:3656-3659 (1980)). In this aspect of the invention, cross-linking occurs at the site in need of augmentation and cross-linking of the polymer can occur intermolecularly and/or to the surrounding tissues, anchoring the composition in place. For example, cysteine can be introduced into the polymer to allow for linkage via disulfide bridges to a surface, or lysine can be introduced for enzymatic linkage to a surface, using a natural extracellular enzyme that cross-links for example, collagens and elastins. Example 2, below, illustrates such a polymer, (GVGVP) 2 GKGVP GVGVP GVGFP GFGFP (SEQ ID NO:10), which contains a Lys(K) residue in a sequence that is a demonstrated substrate for the cross-linking enzyme lysyl oxidase.

Another approach for maintaining the polymer at the injection site involves the inclusion of cell attachment sequences (Nicol, et al., J. Biomed. Mat. Res. 26:393-413 (1992)).

The bioelastic polymers can also optionally have insertions of, for example, single amino acids between monomeric units, substitutions of one amino acid for another in an occasional monomer, or inclusion of different polypentapeptide, polyhexapeptide or polytetrapeptide sequences which can be added either in parallel or in sequence to increase strength in elastic modulus or provide some other desired characteristic. See U.S. Pat. Nos. 4,474,851 and 5,064,430. The resulting polymers are thus properly known as copolymers, as they are formed from different monomeric units.

Considerable variations in the amino acids that are present at various locations in the resulting polymer is also possible as long as the multiple β-turns with intervening suspended bridging segments are retained in order to preserve elasticity. For this reason, it is preferred that at least 50% of the polypeptide is formed from the repeating monomeric units, more preferably at least 70%, even more preferably at least 90%. Nevertheless, it is possible to prepare polypeptides in which these monomeric units are interspersed throughout a larger polypeptide that contains peptide segments designed for other purposes. For example, the bioelastomer can contain naturally occurring sequences which are components of connective tissue or impart functions such as biological activity, chemotaxis, cell targeting and/or adhesion, drug attachment, or protease or nuclease susceptibility. The sequences can be added covalently and sequentially or as side chains to provide for the desired function. The ratio of these other sequences to the monomer residue can range from 1:2 to 1:5000. Preferably the ratio is 1:10 to 1:100. The upper limit on the number and kind of substituents is also influenced by the ability of the elastic polymer to fold/assemble properly to attain a beta-spiral in the relaxed state.

For instance, Example 3 illustrates a protein-based polymer containing a cell attachment sequence, Arg-Gly-Asp, from fibronectin to result in the elastic matrix (GVGVP) 10 GVGVPGRGDSP (GVGVP) 10 (SEQ ID NO:13). Further examples of matrices that promote cell adhesion, cell spreading and growth are X 20 -poly[40(GGAP), (GRGDSP)] (SEQ ID NO:21) and poly[20(GVGVP),(GRGDSP)] (SEQ ID NO:53) (Nicol, et al., “Cell Adhesive Properties of Bioelastic Materials Containing Cell Attachment Sequences,” In Biotechnol. Bioactive Polym, (Charles G. Gebelein and Charles E. Carraher, Jr., Eds.), Plenum Press, New York, pp. 95-113 (1994)). Such polymers allow cells (e.g., fibroblasts) to migrate into and attach to the matrix and to sense the tensional forces to which the matrix is subjected in its functional role, thus allowing the natural cells to regenerate the tissue to sustain the forces required for the tissue to function appropriately. Thus, numerous methods exist, which can be used in combination, to maintain the polymer at the site in need of augmentation and resulting in long term augmentation.

In order to obtain high molecular weight polymers in good yields, a number of approaches are available. Synthesis of the bioelastic repeating units is straightforward and easily accomplished by a peptide chemist or by standard methods in microbial fermentation. For example, organic synthesis of the polymers has been described in the patents listed in the Background Section. In particular, the synthesis and cross-linking of poly(GVGVP) have been described in U.S. Pat. No. 4,783,523. The synthesis of poly(IPGG) has been described in U.S. Pat. No. 5,250,516 and that of poly(GGAP) in U.S. Pat. No. 5,527,610. Accordingly, the teachings of these patents can be applied to the synthesis of bioelastic polymers having different monomer units. When producing polymers by chemical synthesis, care should be taken to avoid impurities, because small levels of impurities can result in termination of the polymerization process or in racemerization that can alter the physical properties of the resulting polymer, but there are otherwise no particular problems of synthesis. Peptide unit purity is important in obtaining a material with suitable physical properties, since small changes in the preparation of the pentamers can result in a transition temperature that varies as much as 15° C. This variance is important to consider since a polymer that has a 25° C. transition temperature will be injectable at 37° C., whereas a preparation having a 40° C. transition temperature may not have the desired properties for tissue augmentation. The solution of this potential problem is simply to purify the components used to prepare the peptide.

Pure or substantially pure bioelastomers of the invention have been found to be biologically inert. However, it has been found that even a less than pure bioelastomer will find utility in the methods of the invention. For example, less then pure (GVGVP) 251 (SEQ ID NO:18) was found to induce a tissue reaction sufficient to form a fibrous capsule, thus maintaining the volume, i.e., insignificant volume change after injection, as is required for the tissue augmentation method of the invention.

The polymer can be prepared as a homopolymer or a copolymer. Either random or block copolymers prepared from at least two of the monomeric units are useful in the methods of the present invention but are less preferred when an equivalent homopolymer has the desired physical properties, simply because of the greater complexity of synthesis. Irrespective of how the bioelastic polymers are synthesized, these can further be derivatized, if desired. For example, photoresponsive or electroresponsive side chains can be incorporated into the polymer as described in U.S. patent application Ser. Nos. 08/187,441 and 08/487,594.

The protein-based polymers can also be prepared using genetic engineering techniques. Using this approach, a gene encoding the desired peptide sequence is constructed, artificially inserted into, and then translated in a host organism. The organism can be prokaryotic, e.g., bacterial, or eukaryotic, e.g., yeast or plants. Techniques are known in the art of molecular biology to manipulate genetic information (i.e., DNA sequences) for effective gene expression in an appropriate host organism (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989)) and include the use of enzymes capable of cleaving, joining, copying or otherwise modifying polynucleotides. In addition, vectors allowing the introduction of this information into the host organism in a suitable manner for expression are known in the art. A detailed example of the production of poly-VPGVG is set out in McPherson, et al., Biotechnol. Prog. 8:347-352 (1992), a publication arising from the laboratory of the present inventor. This publication can be used as guidance for genetic-based production of any of the materials used in the present invention. Polymers with as many as 2000 amino acid residues have been expressed in good quantity by appropriate E. coli strains. For example, expression of (GVGVP) 121 (SEQ ID NO:40) has occurred at levels of 80% of E. coli cell volume. Thus, the cost of production of these protein-based polymers can be competitive with synthetic, organic polymers and with natural materials that need extensive purification. Producers of industrial proteins have demonstrated that costs can be reduced significantly for biologically produced proteins. Of course, medical applications requiring high purity can accept higher costs for medically important applications. For example, costs for the collagen-containing product Contigen®, intended to alleviate urinary incontinence, are understood to be in the range of $1000 (U.S.) per gram.

The bioelastic polypeptide can be purified, for example, from cultures grown in fermentation reactors or from organic syntheses, by its ability to undergo an inverse temperature transition. Purification using the inverse temperature transitional properties of the protein-based polymers is preferred with genetically engineered polymers expressed in microbial systems as endotoxin levels have been demonstrated to be particularly reduced using this method.

The bioelastic polypeptide compositions are biocompatible as a tissue implant and are readily sterilizable. “Biological compatible” as used herein, relates to materials that in final form will not harm the organism or cell into which it is implanted to such a degree that implantation is as harmful or more harmful than the defect being corrected. Biocompatibility can be ascertained by numerous methods known to one of ordinary skill in the art (Picciolo, et al., “A Biotechnology-derived Biomaterials Modulate Host Cell Reactive Oxygen Production as Measured by Chemiluminescence” 19th Annual Meeting, Society for Biomaterials, Birmingham, Ala. (1 993); Urry, et al., Mat. Res. Soc. Symp. Proc. 292:253-264 (1993); and Hoban, et al., J. Surgical Res. 56:179-183 (1984)). For example, it can be determined whether the bioelastic material is capable of eliciting an immune response. Preferably, the bioelastic compositions are non-immunogenic. “Non-immunogenic” as used herein relates to materials which provoke no substantial immune response, inflammation or foreign body reaction when administered. The material can also be tested for biocompatibility by implanting the material in an intended host or substitute test animal and observing the implant at numerous time points after implantation for any adverse reactions.

The bioelastic polypeptide compositions can be selected to be biodegradable. “Biodegradable” as used herein relates to the potential for a material to be degraded by the action of enzymes (including, for example, proteases), or other biological processes, to yield non-toxic substances or byproducts which are compatible with normal body processes. The degree to which a material is biodegradable can vary depending on the particular indication. The propensity of such biopolymers to act innocuously in the body allows these polymers to be injected at sites in need of tissue augmentation where they can reside in the body for days, weeks or longer. Polymer stability can be controlled by appropriate design of the polymer, as it is apparent to one of ordinary skill in the art. For example, protease cleavage sites can be included in the polymer's primary sequence to increase the polymer's susceptibility to protease, or “chemical clocks” as described in European Patent No. 0449592 or its corresponding U.S. application.

Injectable compositions of the invention are useful in plastic surgery, for example, for tissue reconstruction or dermal augmentation (for example, for filling in dermal creases and providing support for skin surfaces), sphincter augmentation (for example, for restoration of urinary continence), delivery of cells, tumor blood vessel blockage, tumor therapy, and contraception/infertility treatments. For example, the problem of stress urinary incontinence can be overcome by adding a periurethral bulking capacity at the base of the bladder. Simply by replacing a Glu (E) residue of a (GVGVP 4 , GEGVP 1 ) (SEQ ID NO:22) polymer by an occasional ArgGlyGlu sequence (from the GRGD SP cell attachment sequence (SEQ ID NO:46)), subcutaneous injection of polymer causes generation or restoration of a natural tissue with the normal distribution of elastic and collagen fibers, rather than simply disappearing. Using the same polymer composition, but cross-linking it to form a matrix with an elastic modulus matching that of the natural bladder and placing on it a human urethral explant containing uroepithelial cells, the cells grow out onto the matrix, but the growth is stimulated toward a greater density of cells and extraceliular matrix by simulating the tensional force changes of bladder filling and emptying.

A particularly useful technique is the replacement of degraded or injured nucleus pulposus in intervertebral discs. The bioelastic polymers described herein can be used in the restoration of diseased intervertebral discs by means of arthroscopic implantation of pressure-responsive and cell stimulation bioelastic materials directly into the depleted nucleus pulposus. In this approach, the prosthesis should match the mechanical properties of the tissue being restored, and, in preferred embodiments, it should contain cell attachment sites to which the normal cells of the tissue attach.

In this embodiment of the invention, the method for tissue restoration of intervertebral discs in a mammal, comprises the step of injecting a polymer into the depleted nucleus pulposus site, which has a site temperature, said polymer comprising repeating peptide monomeric units selected from the group consisting of nonapeptide, pentapeptide and tetrapeptide monomeric units, wherein said monomeric units form a series of β-turns separated by dynamic bridging segments suspended between said β-turns, wherein said polymer has an inverse temperature transition T t less than said site temperature, and wherein said polymer is injected as a water solution at coacervate concentration in the substantial absence of additional water and swells to increase the pressure within the disc.

Typically, during an endoscopic discectomy or other percutaneous procedure, the degraded or injured nucleus pulposus is removed from the intervertebral discs by commercially available endoscopic discectomy equipment. First, a trocar is used to make a hole in the annulus. The endoscopic discectomy equipment is then inserted through the hole and the nucleus pulposus removed. Injection of the bioelastomers of the invention can be injected into the site through the pre-formed hole, either by a large syringe or by modification of the discectomy equipment to provide for loading and delivery of the bioelastomer into the space vacated by the nucleus pulposus.

In another embodiment of the invention, the method for tissue restoration of intervertebral discs in a mammal, comprises the step of injecting a polymer into the depleted nucleus pulposus site, which has a site temperature (T s ), said polymer comprising repeating peptide monomeric units selected from the group consisting of pentapeptide and tetrapeptide monomeric units, alone or in combination, wherein the monomeric units form a series of β-turns separated by dynamic bridging segments suspended between the β-turns, and wherein (i) the polymer has an inverse temperature transition T t less than T s , (ii) the polymer is injected as a water solution at coacervate concentration in the substantial absence of additional water and swells to increase the pressure within the disc, and (iii) the coacervate has an elastic modulus at T s within the range of 5×10 4 to 5×10 6 N/m 2 .

First, the selected bioelastic materials (which are pressure responsive, viscoelastic protein-based polymers) are designed to restore proper dimensions by arthroscopic emplacement in the depleted nucleus pulposus in a state that results in the correct swelling pressure, i.e., they are designed so as to be capable of effecting turgor pressure development on implantation for the purpose of mechanical restoration of the compressed disc to its correct dimensions and internal pressure. Second, biologically active sequences can optionally (and preferably) be included in the sequence to induce tissue regeneration or restoration while the disc is in an improved structural/functional state, i.e., they are designed so as to be capable of stimulating tissue restoration of the intervertebral disc to its more natural state. By multiple attachments of cell to the mechanically correct matrix, which acts as a temporary functional scaffolding, the mechanical stresses sustained by the functional artificial matrix would be appropriately transmitted to the cells. Since it is believed that cells are mechano-chemical transducers that respond to the force changes that they sense, this mechanical sensing by the cells would result in the chemical remodeling of the matrix into a natural tissue sufficient to sustain those forces (that is, in the elaboration of the required extracellular matrix). Polymers of particular interest for this application include, by way of illustration and not limitation, (GVGIP) n (SEQ ID NO:51) and [(GVGIP) 10 ] n (SEQ ID NO:56), along with [(GVGVP) 2 GKGVP GVGFP GFGFP] n (SEQ ID NO:54), [(GVGIP) 10 GVGVPGRGDSP (GVGIP) 10 ] n (SEQ ID NO:57), [(GVGIP) 10 GVGVPGRGDSP(GVGVP) 10 ] n (SEQ ID NO:58), [{(GVGVP) 2 GFGVP} 3 ] n (SEQ ID NO:59) and [(GVGIP) 10 GVGVPGRGDSP (GVGVP GVGVP GKGVP GVGFP GFGFP) 2 ] n (SEQ ID NO:60).

As noted earlier, the value of T t depends upon numerous factors and, accordingly, the value of T t can be shifted to carry out a wide variety of energy conversions, including the energy required to maintain an internal pressure for an intervertebral disc.

As relates to tensional force changes, this mechano-chemical transductional property of cells has been referred to as “cellular tensegrity”. (Ingber, Int. Rev. Cytol. 150:173-224 (1994) and Ingber, J. Cell Sci. 104:613-627 (1993)). It is a more general concept, however, that includes both compressional and tensional forces. Recent studies have shown that the cells of the intervertebral disc are mechano-chemical transducers and the proper mechanical energy input in terms of applied pressures and the time course of fluctuation in pressure dictate chemical output of the cells of the intervertebral disc (Ishihara, et al., J. Appl. Physiol. 80(3):839-846 (1996)).

Another key aspect is the ability of diseased discs to maintain hydrostatic pressures developed on emplacement of the hygroscopic viscoelastic protein based polymers. Studies on autopsy specimens indicate that the nucleus pulposus behaves hydrostatically as long as the disc has not undergone extensive degeneration. (Nachemson, et al., J. Bone Jt. Surg. Am. 46A:1077-1092 (1964) and Nachemson, Acta Orthop. Scandinavica , Suppl. 43:1-104 (1960)). The outer tendinous bands of the annulus fibrosis come under increased tension on increasing hydrostatic pressure whereas those of the inner annulus come under compression. Further, examination of the compressive stress has shown that intervertebral discs behave as fluid filled pressure vessels (Nachemson, et al., J. Bone Jt. Surg. Am. 46A:1077-1092 (1964)). These two observations factor into the approach taken to design protein-based polymers for intervertebral disc restoration.

There are two pressure sensitive aspects of the viscoelastic proteins based polymers described herein that are important in the design and/or selection of polymers useful for the restoration of intervertebral discs. One is demonstrated by (GVGIP) 260 (SEQ ID NO:55) and the other by the introduction of pressure sensitivity by T t by the presence of aromatic residues. This is explained in greater detail in the examples.

Additional biologically important factors may be added for repair of hard tissue (for example, mineral additives for correction of bone defects or osteogenic factors including bone morphogenic proteins to promote mineralization or bone formation) or to enhance cell growth in the vicinity of the injection (for example, cell-growth or cell-attraction agents, host-compatible cells or growth factors). The appropriate amount of material to be added to the composition to achieve the desired effect will be apparent to the medical practitioner and is not a requirement of the present invention, as such selection is usually made by the physician (aided, if necessary, by analytical analysis of biopsied tissue) to achieve the result desired by the physician and/or required by the injury or defect. Such factors are supplemental to the invention itself and are optional, not being required or desirable in many cases.

The bioelastic material to be injected can be dissolved or suspended in a pharmaceutically acceptable liquid carrier prior to injection. Examples of an appropriate liquid include physiologically buffered salt solutions, water, glycerol and the like, and may be supplemented with, for example, serum, growth factors, hormones, sugars, amino acids, vitamins, metalloproteins, lipoproteins, and the like. Most of the carrier will be removed prior to injection, as discussed above, but some may remain in the final injectable composition. Thus, compositions used in the invention may additionally include one or more biologically active factors to aid in the healing or regrowth of natural tissue. For example, one may incorporate factors such as heparin, epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), TGF-β, platelet-derived growth factor, fibroblast growth factor, connective tissue activating peptides, β-thromboglobulin, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, osteogenic factors including bone morphogenic proteins, and the like. Incorporation of such factors, and appropriate combinations of factors, can facilitate the tissue treatment. Decisions to use such factors are typically made by the attending physician based on judgments about the injury or defect being repaired.

Similarly, one can inject a mixture comprising cells and the bioelastic polymer at the site in need of augmentation. The cells are living cells, preferably from the host receiving the injection or from a cell source sufficiently related to avoid graft/host interactions leading to rejection, including human cells. Cells can be autogeneic, isogeneic, allogeneic or xenogeneic, more preferably autogeneic or allogeneic. Included are cells which have been genetically engineered. The bioelastic compositions can contain different cell types, which may be chosen to act synergistically, for example, in the formation of tissue. Examples of types of cells include muscle cells, nerve cells, epithelial cells, connective tissue cells, and organ cells. Specific examples of cells include fibroblast cells, smooth muscle cells, striated muscle cells, heart muscle cells, nerve cells, epithelial cells, endothelial cells, bone cells, bone progenitor cells, bone marrow cells, blood cells, brain cells, kidney cells, liver cells, lung cells, pancreatic cells, spleen cells, breast cells, foreskin cells, ovary cells, testes cells and prostate cells. Other mammalian cells are useful in the practice of the invention and are not excluded from consideration here. Alternatively, the injectable bioelastic compositions can include non-mammalian eukaryotic cells, prokaryotic cells or viruses.

The present invention also provides kits for performing the method of the invention. Such kits can be prepared from readily available materials and reagents and can come in a variety of embodiments. For example, such kits can comprise, in an amount sufficient for at least one treatment, any one or more of the following materials: bioelastic polymer, vessels, sterilized buffers (e.g., phosphate buffered saline) or water, other reagents necessary or helpful to perform the method, and instructions. Typically, instructions include a tangible expression describing reagent concentration or at least one method parameter, such as the amount of reagent to be used, maintenance time periods for reagents, and the like, to allow the user to carry out the methods described above. In a preferred embodiment of the invention, a kit comprises a means for delivery in which is placed the bioelastic polymer, both of which are often pre-sterilized. Such means can include, by way of illustration and not limitation, a small syringe (22 to 27-gauge), a large syringe (13 to 19-gauge) and equipment used in endoscopic or percutaneous discectomy procedures. The reagents can be provided in solution, as suspensions, or as a substantially dry powder, e.g., in lyophilized form, either independently or in a mixture of components to improve ease of use. Where a degradable reagent is provided, conditions are chosen so as to stabilize the reagent, e.g., storage at lower temperature, addition of stabilizing agents (e.g., glycerol or a reducing agent). Unstable reagents can be provided together with or separately from the more stable components of the kit.

A particularly preferred embodiment of a kit is a kit for augmentation of a tissue, the tissue having a normal tissue temperature, comprising a syringe (either small or large), a sterile wrapper surrounding the syringe and providing a sterile environment for the syringe, and a polymer contained in the syringe, wherein the polymer comprises repeating peptide monomeric units selected from the group consisting of nonapeptide, pentapeptide and tetrapeptide monomeric units, wherein the monomeric units form a series of β-turns separated by dynamic bridging segments suspended between the β-turns, and wherein the polymer has an inverse temperature transition T t less than the tissue temperature. The polymer can be present in the syringe either in solution or as a water solution at coacervate concentration in the substantial absence of additional water. Where the polymer is present in solution, the syringe and the polymer it contains are maintained at low temperatures to stabilize the polymer, since the polymer is usually in solution rather than in the form of a coacervate at low temperatures (depending, as discussed above, on the value of T t ). In such cases the coacervate is formed by raising the temperature of the syringe and polymer to above T t , after which the excess water (now in a separate phase from the coacervate) can be expelled from the syringe prior to injection of the coacervate. An example of such use is described below.

The following examples describe specific aspects of the invention to illustrate the invention and provide a description of the present method for those of skill in the art. The examples should not be construed as limiting the invention, as the examples merely provide specific methodology useful in understanding and practice of the invention.

The preparation of the elastic protein-based polymers utilizes gene construction, development of an expression system, fermentation and purification as described for the particular examples given below. As for gene construction, the basic monomer genes were designed to have appropriate cohesive ends comprising an appropriate restriction site sequence (selected using standard techniques, such as consideration of the restriction sites present in the vector to be used for expression). Polymerization of the basic gene was carried out through the compatible sticky ends generated by restriction endonuclease digestion, followed by subsequent ligation using DNA ligase to form multimers of the basic gene. This protocol was used successfully to produce each of the monomer gene sequences. The monomers were then concatamerized (polymerized) to form multimer genes with many different numbers of repeats, and many of the multimer genes have been expressed at high levels as will be briefly reported below.

SEQ ID NO:1 depicts a 5′ oligomer construct for producing polymer units of SEQ ID NO:9.

SEQ ID NO:2 depicts a 3′ oligomer construct for producing polymer units of SEQ ID NO:10 and polymer II (SEQ ID NO:36).

SEQ ID NO:3 and 4 depict 3′ oligomer constructs for producing polymer units of SEQ ID NO:11.

SEQ ID NO:5 depicts a 3′ oligomer construct for producing polymer units of SEQ ID No. 12 and polymer I (SEQ ID NO:35).

SEQ ID NO:6 is a synthetic gene sequence coding for SEQ ID No. 13 and polymer III (SEQ ID NO:37).

SEQ ID NO:7 is a synthetic gene sequence coding for SEQ ID No. 14 and polymer IV (SEQ ID NO:38).

SEQ ID NO:8 is a synthetic gene sequence coding for SEQ ID No. 15 and polymer V (SEQ ID NO:39).

SEQ ID NO:9 is the protein sequence of a tetrameric elastomer.

SEQ ID NO:10 is the protein sequence of a lysine and phenylalanine containing elastomer.

SEQ ID NO:11 is the protein sequence of GRGDSP cell adhesion containing sequence.

SEQ ID NO:12 is the protein sequence of a lysine containing elastomer.

SEQ ID NO:13 is the protein sequence of a GRGDSP containing elastomer.

SEQ ID NO:14 is the protein sequence of an alanine containing elastomer.

SEQ ID NO:15 is the protein sequence of a glutamic acid containing elastomer.

SEQ ID NO:16 and 17 are tetrapeptide and pentapeptide monomers, respectively.

SEQ ID NO:18 is a polypentapeptide elastomer.

SEQ ID NO:19 is a pentapeptide monomer.

SEQ ID NO:20 is a polymer comprised of pentapeptide monomers.

SEQ ID NO:21 is the protein sequence of a GRGDSP containing elastomer.

SEQ ID NO:22 is the protein sequence of a glutamic acid containing elastomer.

SEQ ID NO:23 and 24 are oligonucleotides for producing a phenylalanine containing elastomer (SEQ ID NO:9).

SEQ ID NO:25 is the protein sequence of a lysine and phenylalanine containing elastomer.

SEQ ID NO:26 and 27 are oligonucleotides for producing a lysine and phenylalanine containing elastomer (SEQ ID NO:10).

SEQ ID NO:28 is the protein sequence of the cell adhesion sequence.

SEQ ID NO:29 is the protein sequence of an elastomer comprised of pentapeptide monomers.

SEQ ID NOS:30, 31, 32 and 33 are synthetic gene sequences coding for adapter oligomers used to clone concatamers of the various elastomer genes.

SEQ ID NO:34 is the protein sequence of a GRGDSP containing elastomer.

SEQ ID NO:35 is the protein sequence of polymer I, which contains Lys.

SEQ ID NO:36 is the protein sequence of polymer II, which contains Phe and Lys.

SEQ ID NO:37 is the protein sequence of polymer III, which contains a cell adhesion sequence.

SEQ ID NO:38 is the protein sequence of polymer IV

SEQ ID NO:39 is the protein sequence of polymer V, which contains Glu.

SEQ ID NO:40 is a polypentapeptide elastomer.

SEQ ID NOS:41 and 42 are tetrapeptide monomers.

SEQ ID NOS:43, 44 and 45 are pentapeptide monomers.

SEQ ID NO:46 is the GRGDSP cell attachment sequence.

SEQ ID NO:47 is GVGVAP.

SEQ ID NOS:48 and 49 are pentapeptide monomers.

SEQ ID NO:50 is a tetrapeptide monomer.

SEQ ID NO:51 is a pentapeptide monomer.

SEQ ID NO:52 is VGVAPG.

SEQ ID NO:53 is the protein sequence of a GRGDSP containing elastomer.

SEQ ID NOS:54, 55 and 56 are protein sequences of elastomers comprised of pentapeptide monomers, where SEQ ID NO:54 is polymer VII and SEQ ID NO:56 is polymer VI.

SEQ ID NOS:57 and 58 are protein sequences of GRGDSP containing elastomers, polymers VIII and IX, respectively.

SEQ ID NO:59 is the protein sequence of an elastomer comprised of pentapeptide monomers, polymer X.

SEQ ID NO:60 is the protein sequence of a GRGDSP containing elastomer, polymer XI.

SEQ ID NO:61 is the protein sequence of an elastomer comprised of pentapeptide monomers.

SEQ ID NO:62 is the protein sequence of a fragment of polymer XI.

SEQ ID NOS:63 and 64 are synthetic gene sequences coding for adapter oligomers used to clone the gene for polymer XI.

SEQ ID NO:65 is the protein sequence of a fragment of polymer XI.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference at the location where cited.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention.

1

gaggatccga agacaacagg tggtgttccg ggcggcgtac cgggtggcgt accgggcggt 60

ttcccgggag gtgtgccggg tggggttcca ggcggtgtac cgggtgggtt tccgggcggt 120

gttccgggtg gagttccggg tggcgtgccg ggcggttttc caggaagtct tcggatccag 180

2

gaggatccag gcgttggggt accgggtgtt ggcgtaccgg gtaaaggtgt cccgggcgtt 60

ggtgtgccgg gtgtaggctt tccgggtttc ggattcccag gcgttggatc cag 113

3

taggggtacc gggtcgtggt gactctccgg gcg 33

4

cgcatcccca tggcccagca ccactgagag gcc 33

5

gaggatccag gcgttggggt accgggtgtt ggcgtaccgg gtgttggtgt cccgggcaaa 60

ggtgtgccgg gtgtaggcgt tccgggtgtg ggagtcccag gcgttggatc c 111

6

ggcgttggtg taccgggtgt tggtgtgccg ggtgttggtg ttccgggcgt aggcgtaccg 60

ggcgtaggcg tgccgggcgt aggcgttccg ggcgtgggcg taccgggcgt gggcgtgccg 120

ggtgtgggcg tcccgggtgt aggtgttcca ggcgtagggg taccgggtcg tggtgactct 180

ccgggcgttg gtgtaccggg tgttggtgtg ccgggtgttg gtgttccggg cgtaggcgta 240

ccgggcgtag gcgtgccggg cgtaggcgtt ccgggcgtgg gcgtaccggg cgtgggcgtg 300

ccgggtgtgg gcgtcccggg tgtaggtgtt ccaggcgttg gatcc 345

7

ggatccaggc gttggtgtac cgggtgttgg tgtgccgggt gttggtgttc cgggcgtagg 60

cgtaccgggc gtaggcgtgc cgggcgtagg cgttccgggc gtgggcgtac cgggcgtggg 120

cgtgccgggt gtgggcgtcc caggtgtagg cgttccgggt gtgggtgtag ctccgggtgt 180

tggcgttgca ccgggcgtag gtgttgctcc gggcgttggc gtggcgccgg gtgttggtgt 240

tgctccgggt gtaggcgttg ctccgggcgt tggtgttgcc ccaggtgtag gtgtggcacc 300

gggcgttggt gtaccgggtg ttggtgtgcc gggtgttggt gttccgggcg taggcgtacc 360

gggcgtaggc gtgccgggcg taggcgttcc gggcgtgggc gtaccgggcg tgggcgtgcc 420

gggtgtgggc gtcccgggtg taggtgttcc aggcgttgga tcc 463

8

gaggatccag gcgttggggt accgggtgtt ggcgtaccgg gtgaaggtgt cccgggcgtt 60

ggtgtgccgg gtgtaggcgt tccgggtgtg ggagtcccag gcgttggatc c 111

9

Gly Gly Val Pro Gly Gly Val Pro Gly Gly Val Pro Gly Gly Phe Pro

1 5 10 15

Gly Gly Val Pro Gly Gly Val Pro Gly Gly Val Pro Gly Gly Phe Pro

20 25 30

Gly Gly Val Pro Gly Gly Val Pro Gly Gly Val Pro Gly Gly Phe Pro

35 40 45

10

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro

20 25 30

11

Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly

1 5 10

12

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

20 25 30

13

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly

50 55 60

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

65 70 75 80

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

85 90 95

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

100 105 110

14

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val

50 55 60

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro

65 70 75 80

Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

85 90 95

Ala Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

100 105 110

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

115 120 125

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

130 135 140

Val Gly Val Pro

145

15

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

20 25 30

16

Val Pro Gly Gly

1

17

Val Pro Gly Val Gly

1 5

18

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

65 70 75 80

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

100 105 110

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

115 120 125

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

130 135 140

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

145 150 155 160

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

165 170 175

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

180 185 190

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

195 200 205

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

210 215 220

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

225 230 235 240

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

245 250 255

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

260 265 270

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

275 280 285

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

290 295 300

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

305 310 315 320

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

325 330 335

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

340 345 350

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

355 360 365

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

370 375 380

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

385 390 395 400

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

405 410 415

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

420 425 430

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

435 440 445

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

450 455 460

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

465 470 475 480

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

485 490 495

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

515 520 525

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

545 550 555 560

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

610 615 620

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

625 630 635 640

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

645 650 655

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

660 665 670

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

675 680 685

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

690 695 700

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

705 710 715 720

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

725 730 735

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

740 745 750

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

755 760 765

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

770 775 780

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

785 790 795 800

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

805 810 815

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

820 825 830

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

835 840 845

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

850 855 860

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

865 870 875 880

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

885 890 895

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

900 905 910

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

915 920 925

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

930 935 940

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

945 950 955 960

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

965 970 975

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

980 985 990

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

995 1000 1005

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1010 1015 1020

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1025 1030 1035

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1040 1045 1050

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1055 1060 1065

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1070 1075 1080

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1085 1090 1095

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1100 1105 1110

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1115 1120 1125

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1130 1135 1140

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1145 1150 1155

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1160 1165 1170

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1175 1180 1185

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1190 1195 1200

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1205 1210 1215

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1220 1225 1230

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1235 1240 1245

Val Pro Gly Val Gly Val Pro

1250 1255

amino acid that is modified to have an

electroresponsive side chain

19

Val Pro Gly Xaa Gly

1 5

20

Gly Val Gly Val Pro

1 5

21

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

1 5 10 15

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

20 25 30

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

35 40 45

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

50 55 60

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

65 70 75 80

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

85 90 95

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

100 105 110

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

115 120 125

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

130 135 140

Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro Gly Gly Ala Pro

145 150 155 160

Gly Arg Gly Asp Ser Pro

22

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Glu Gly Val Pro

20 25

23

Gly Ala Gly Gly Ala Thr Cys Cys Gly Ala Ala Gly Ala Cys Ala Ala

1 5 10 15

Cys Ala Gly Gly Thr Gly Gly Thr Gly Thr Thr Cys Cys Gly Gly Gly

20 25 30

Cys Gly Gly Cys Gly Thr Ala Cys Cys Gly Gly Gly Thr Gly Gly Cys

35 40 45

Gly Thr Ala Cys Cys Gly Gly Gly Cys Gly Gly Thr Thr Thr Cys Cys

50 55 60

Cys Gly Gly Gly Ala Gly Gly Thr Gly Thr Gly Cys Cys Gly Gly Gly

65 70 75 80

Thr Gly Gly Gly Gly Thr Thr Cys Cys Ala Gly Gly Cys Gly Gly Thr

85 90 95

Gly Thr Ala Cys

100

24

ctggatccga agacttcctg gaaaaccgcc cggcacgcca cccggaactc cacccggaac 60

accgcccgga aacccacccg gtacaccgcc tggaacccca 100

25

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro

65 70 75 80

Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val

100 105 110

Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly

115 120 125

Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe

130 135 140

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

145 150 155 160

Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly

165 170 175

Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys

180 185 190

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly

195 200 205

Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val

210 215 220

Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro

225 230 235 240

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

245 250 255

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val

260 265 270

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

275 280 285

Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val

290 295 300

Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro

305 310 315 320

Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly

325 330 335

Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val

340 345 350

Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly

355 360 365

Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe

370 375 380

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

385 390 395 400

Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly

405 410 415

Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys

420 425 430

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly

435 440 445

Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val

450 455 460

Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro

465 470 475 480

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

485 490 495

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

515 520 525

Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro

545 550 555 560

Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590

Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe

610 615 620

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro

625 630 635

26

gaggatccag gcgttggggt accgggtgtt ggcgatccgg gtaaaggtgt cccggggttg 60

gtgtgc 66

27

ctggatccaa cgcctgggaa tccgaaaccc ggaaagccta cacccggcac accaacgccc 60

gggaca 66

28

Gly Arg Gly Asp Ser Pro

1 5

29

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro

50

30

ctggatccag accatgggcg tt 22

31

ggcgttggtg taccgtaagc ttgaattcgg atccag 36

32

gacctaggtc tggtacccgc aa 22

33

ccgcaaccac atggcattcg aacttaagcc taggtc 36

34

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly

50 55 60

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

65 70 75 80

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

85 90 95

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

100 105 110

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

115 120 125

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

130 135 140

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

145 150 155 160

Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val

165 170 175

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

180 185 190

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

195 200 205

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

210 215 220

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

225 230 235 240

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

245 250 255

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

260 265 270

Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro

275 280 285

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

290 295 300

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

305 310 315 320

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

325 330 335

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

340 345 350

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

355 360 365

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

370 375 380

Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly

385 390 395 400

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

405 410 415

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

420 425 430

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

435 440 445

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

450 455 460

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

465 470 475 480

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

485 490 495

Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

515 520 525

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

545 550 555 560

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly

610 615 620

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

625 630 635 640

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

645 650 655

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

660 665 670

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

675 680 685

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

690 695 700

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

705 710 715 720

Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly Val

725 730 735

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

740 745 750

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

755 760 765

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

770 775 780

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

785 790 795 800

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

805 810 815

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

820 825 830

Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

835 840 845

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

850 855 860

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

865 870 875 880

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

885 890 895

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

900 905 910

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

915 920 925

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

930 935 940

Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

945 950 955 960

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

965 970 975

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

980 985 990

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

995 1000 1005

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1010 1015 1020

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1025 1030 1035

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1040 1045 1050

Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly

1055 1060 1065

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1070 1075 1080

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1085 1090 1095

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1100 1105 1110

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1115 1120 1125

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1130 1135 1140

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1145 1150 1155

Val Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val

1160 1165 1170

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1175 1180 1185

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1190 1195 1200

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1205 1210 1215

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1220 1225 1230

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1235 1240 1245

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1250 1255 1260

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Arg

1265 1270 1275

Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1280 1285 1290

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1295 1300 1305

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1310 1315 1320

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1325 1330 1335

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1340 1345 1350

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1355 1360 1365

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1370 1375 1380

Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro

1385 1390 1395

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1400 1405 1410

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1415 1420 1425

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1430 1435 1440

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1445 1450 1455

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1460 1465 1470

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1475 1480 1485

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser

1490 1495 1500

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1505 1510 1515

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1520 1525 1530

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1535 1540 1545

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1550 1555 1560

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1565 1570 1575

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1580 1585 1590

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

1595 1600 1605

Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly

1610 1615 1620

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1625 1630 1635

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1640 1645 1650

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1655 1660 1665

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1670 1675 1680

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1685 1690 1695

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1700 1705 1710

Val Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val

1715 1720 1725

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1730 1735 1740

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1745 1750 1755

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1760 1765 1770

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1775 1780 1785

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1790 1795 1800

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

1805 1810 1815

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Arg

1820 1825 1830

Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1835 1840 1845

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1850 1855 1860

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1865 1870 1875

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1880 1885 1890

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1895 1900 1905

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1910 1915 1920

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1925 1930 1935

Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro

1940 1945 1950

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1955 1960 1965

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1970 1975 1980

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

1985 1990 1995

Gly Val Gly Val Pro

2000

35

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro

65 70 75 80

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

100 105 110

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

115 120 125

Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val

130 135 140

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

145 150 155 160

Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

165 170 175

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

180 185 190

Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

195 200 205

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

210 215 220

Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

225 230 235 240

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

245 250 255

Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

260 265 270

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

275 280 285

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

290 295 300

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro

305 310 315 320

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

325 330 335

Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

340 345 350

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

355 360 365

Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val

370 375 380

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

385 390 395 400

Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

405 410 415

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

420 425 430

Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

435 440 445

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

450 455 460

Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

465 470 475 480

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

485 490 495

Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

515 520 525

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro

545 550 555 560

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

580 585 590

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val

610 615 620

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

625 630 635 640

Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

645 650 655

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

660 665 670

Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

675 680 685

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

690 695 700

Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

705 710 715 720

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

725 730 735

Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

740 745 750

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

755 760 765

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

770 775 780

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro

785 790 795 800

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

805 810 815

Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val

820 825 830

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

835 840 845

Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val

850 855 860

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

865 870 875 880

Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly

885 890 895

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

900 905 910

Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

915 920 925

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

930 935 940

Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

945 950 955 960

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

965 970 975

Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

980 985 990

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

995 1000 1005

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1010 1015 1020

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

1025 1030 1035

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1040 1045 1050

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly

1055 1060 1065

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1070 1075 1080

Val Pro

1085

36

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro

65 70 75 80

Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val

100 105 110

Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly

115 120 125

Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe

130 135 140

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

145 150 155 160

Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly

165 170 175

Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys

180 185 190

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly

195 200 205

Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val

210 215 220

Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro

225 230 235 240

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

245 250 255

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val

260 265 270

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

275 280 285

Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val

290 295 300

Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro

305 310 315 320

Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly

325 330 335

Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val

340 345 350

Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly

355 360 365

Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe

370 375 380

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

385 390 395 400

Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly

405 410 415

Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys

420 425 430

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly

435 440 445

Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val

450 455 460

Pro Gly Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro

465 470 475 480

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

485 490 495

Val Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly

515 520 525

Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro

545 550 555 560

Gly Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590

Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Lys Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Phe

610 615 620

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro

625 630 635

37

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly

50 55 60

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

65 70 75 80

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

85 90 95

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

100 105 110

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

115 120 125

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

130 135 140

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

145 150 155 160

Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val

165 170 175

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

180 185 190

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

195 200 205

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

210 215 220

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

225 230 235 240

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

245 250 255

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

260 265 270

Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro

275 280 285

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

290 295 300

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

305 310 315 320

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

325 330 335

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

340 345 350

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

355 360 365

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

370 375 380

Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly

385 390 395 400

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

405 410 415

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

420 425 430

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

435 440 445

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

450 455 460

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

465 470 475 480

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

485 490 495

Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

515 520 525

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

545 550 555 560

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly

610 615 620

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

625 630 635 640

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

645 650 655

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

660 665 670

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

675 680 685

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

690 695 700

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

705 710 715 720

Pro Gly Arg Gly Asp Ser Pro Gly Val Gly Val Pro Gly Val Gly Val

725 730 735

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

740 745 750

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

755 760 765

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

770 775 780

38

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val

50 55 60

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro

65 70 75 80

Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

85 90 95

Ala Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

100 105 110

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

115 120 125

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

130 135 140

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

145 150 155 160

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

165 170 175

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

180 185 190

Pro Gly Val Gly Val Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

195 200 205

Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val

210 215 220

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro

225 230 235 240

Gly Val Gly Val Ala Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

245 250 255

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

260 265 270

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

275 280 285

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

290 295 300

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

305 310 315 320

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

325 330 335

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Ala Pro

340 345 350

Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

355 360 365

Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val

370 375 380

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Pro Gly

385 390 395 400

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

405 410 415

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

420 425 430

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

435 440 445

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

450 455 460

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

465 470 475 480

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

485 490 495

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro

500 505 510

Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

515 520 525

Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val

530 535 540

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

545 550 555 560

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

565 570 575

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

580 585 590

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

595 600 605

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

610 615 620

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

625 630 635 640

Val Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val

645 650 655

Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro

660 665 670

Gly Val Gly Val Ala Pro Gly Val Gly Val Ala Pro Gly Val Gly Val

675 680 685

Ala Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

690 695 700

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

705 710 715 720

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

725 730 735

Val Gly Val Pro Gly Val Gly Val Pro

740 745

39

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro

65 70 75 80

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val

100 105 110

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

115 120 125

Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val

130 135 140

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

145 150 155 160

Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly

165 170 175

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

180 185 190

Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

195 200 205

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

210 215 220

Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

225 230 235 240

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

245 250 255

Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

260 265 270

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

275 280 285

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

290 295 300

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro

305 310 315 320

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

325 330 335

Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val

340 345 350

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

355 360 365

Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val

370 375 380

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

385 390 395 400

Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly

405 410 415

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

420 425 430

Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

435 440 445

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

450 455 460

Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

465 470 475 480

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

485 490 495

Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

515 520 525

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro

545 550 555 560

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val

580 585 590

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

595 600 605

Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val

610 615 620

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

625 630 635 640

Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly

645 650 655

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

660 665 670

Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

675 680 685

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

690 695 700

Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

705 710 715 720

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

725 730 735

Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

740 745 750

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

755 760 765

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

770 775 780

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro

785 790 795 800

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

805 810 815

Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val

820 825 830

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

835 840 845

Val Pro Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val

850 855 860

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

865 870 875 880

Gly Val Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly

885 890 895

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

900 905 910

Gly Val Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

915 920 925

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

930 935 940

Pro Gly Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

945 950 955 960

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

965 970 975

Glu Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

980 985 990

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

995 1000 1005

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1010 1015 1020

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

1025 1030 1035

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1040 1045 1050

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Glu Gly

1055 1060 1065

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

1070 1075 1080

Val Pro

1085

40

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

65 70 75 80

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

100 105 110

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

115 120 125

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

130 135 140

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

145 150 155 160

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

165 170 175

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

180 185 190

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

195 200 205

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

210 215 220

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

225 230 235 240

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

245 250 255

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

260 265 270

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

275 280 285

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

290 295 300

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

305 310 315 320

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

325 330 335

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

340 345 350

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

355 360 365

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

370 375 380

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

385 390 395 400

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

405 410 415

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

420 425 430

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

435 440 445

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

450 455 460

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

465 470 475 480

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

485 490 495

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

500 505 510

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

515 520 525

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

530 535 540

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

545 550 555 560

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

565 570 575

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

580 585 590

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

595 600 605

41

Gly Gly Val Pro

1

42

Gly Gly Phe Pro

1

43

Gly Lys Gly Val Pro

1 5

44

Gly Val Gly Phe Pro

1 5

45

Gly Phe Gly Phe Pro

1 5

46

Gly Arg Gly Asp Ser Pro

1 5

47

Gly Val Gly Val Ala Pro

1 5

48

Gly Glu Gly Val Pro

1 5

49

Gly Phe Gly Val Pro

1 5

50

Gly Gly Ala Pro

1

51

Gly Val Gly Ile Pro

1 5

52

Val Gly Val Ala Pro Gly

1 5

53

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly

35 40 45

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

50 55 60

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

65 70 75 80

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

85 90 95

Val Gly Val Pro Gly Arg Gly Asp Ser Pro

100 105

54

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

1 5 10 15

Val Gly Phe Pro Gly Phe Gly Phe Pro

20 25

55

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

1 5 10 15

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

20 25 30

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

35 40 45

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

50 55 60

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

65 70 75 80

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

85 90 95

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

100 105 110

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

115 120 125

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

130 135 140

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

145 150 155 160

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

165 170 175

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

180 185 190

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

195 200 205

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

210 215 220

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

225 230 235 240

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

245 250 255

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

260 265 270

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

275 280 285

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

290 295 300

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

305 310 315 320

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

325 330 335

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

340 345 350

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

355 360 365

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

370 375 380

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

385 390 395 400

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

405 410 415

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

420 425 430

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

435 440 445

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

450 455 460

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

465 470 475 480

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

485 490 495

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

500 505 510

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

515 520 525

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

530 535 540

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

545 550 555 560

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

565 570 575

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

580 585 590

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

595 600 605

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

610 615 620

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

625 630 635 640

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

645 650 655

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

660 665 670

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

675 680 685

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

690 695 700

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

705 710 715 720

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

725 730 735

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

740 745 750

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

755 760 765

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

770 775 780

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

785 790 795 800

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

805 810 815

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

820 825 830

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

835 840 845

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

850 855 860

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

865 870 875 880

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

885 890 895

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

900 905 910

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

915 920 925

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

930 935 940

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

945 950 955 960

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

965 970 975

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

980 985 990

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

995 1000 1005

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1010 1015 1020

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1025 1030 1035

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1040 1045 1050

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1055 1060 1065

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1070 1075 1080

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1085 1090 1095

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1100 1105 1110

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1115 1120 1125

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1130 1135 1140

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1145 1150 1155

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1160 1165 1170

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1175 1180 1185

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1190 1195 1200

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1205 1210 1215

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1220 1225 1230

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1235 1240 1245

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1250 1255 1260

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1265 1270 1275

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

1280 1285 1290

Ile Pro Gly Val Gly Ile Pro

1295 1300

56

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

1 5 10 15

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

20 25 30

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

35 40 45

Ile Pro

50

57

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

1 5 10 15

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

20 25 30

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

35 40 45

Ile Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly

50 55 60

Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile

65 70 75 80

Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

85 90 95

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro

100 105 110

58

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

1 5 10 15

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

20 25 30

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

35 40 45

Ile Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly

50 55 60

Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val

65 70 75 80

Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

85 90 95

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

100 105 110

59

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Phe Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Val Gly Val Pro Gly Phe Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Val Gly Val Pro Gly Phe Gly Val Pro

35 40 45

60

Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly

1 5 10 15

Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val

20 25 30

Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly Ile Pro Gly Val Gly

35 40 45

Ile Pro Gly Val Gly Val Pro Gly Arg Gly Asp Ser Pro Gly Val Gly

50 55 60

Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Phe

65 70 75 80

Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val Gly Val Pro

85 90 95

Gly Lys Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly Phe Pro

100 105 110

61

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly

1 5 10 15

Val Gly Val Pro Gly Lys Gly Val Pro

20 25

62

Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Lys Gly Val Pro Gly

1 5 10 15

Val Gly Phe Pro Gly Phe Gly Phe Pro Gly Val Gly Val Pro Gly Val

20 25 30

Gly Val Pro Gly Lys Gly Val Pro Gly Val Gly Phe Pro Gly Phe Gly

35 40 45

Phe Pro

50

63

ttcggattcc cgggcgtagg cgtaccgggt 30

64

aagcctaagg gcccgcatcc gcatggccca 30

65

Phe Gly Phe Pro Gly Val Gly Val Pro Gly

1 5 10